Recombinant Human Anti-HCV E2 Antibody (hu5B3.v3) (CAT#: PABZ-181)

Figure 1 HCVpp neutralization of affinity-matured E2⁴¹²⁻⁴³²-specific antibodies

Pantua, H., Diao, J., Ultsch, M., Hazen, M., Mathieu, M., McCutcheon, K., ... & Hass, P. (2013). Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies. Journal of molecular biology, 425(11), 1899-1914.

Figure 2 Identification and characterization of mutant Jc1 and Con1/C3-neo HCVcc resistant to E2^412–423-specific neutralizing antibody (AP33).

(a) Supernatants and cells from Jc1 (squares) and Con1/C3-neo (circles) HCVcc-infected Huh7.5 cells were sequentially cultured in the absence (filled symbols) or presence (open symbols) of increasing concentrations of AP33, and (b) HCV RNA replication was measured at various times post infection. These are representative data from three independent experiments. (c) 454 Ultra deep sequencing analysis of N417 amino acid variations over time demonstrating an enrichment of the N417S mutant HCVcc when cultured in the presence of antibody (WT, black; N417S, red; N417T, orange; N417G, green). (d) The effect of the mutations on HCVcc infectivity was determined by introducing them in Jc1 HCVcc and monitoring growth in culture over 9 days. (e and f) The effect of the identified mutations on infectivity of HCVpp was determined. HCVpp stocks were generated and normalized using p24 levels in the supernatants (e) followed by infecting Huh7.5 cells to measure infectivity (f). No pseudoparticles were generated in the absence of E1E2-expressing plasmid as determined by p24 levels. In vitro growth and infectivity studies are representative data from at least two independent experiments. p values: *p < 0.05, **p < 0.01 and ***p < 0.001.

Pantua, H., Diao, J., Ultsch, M., Hazen, M., Mathieu, M., McCutcheon, K., ... & Hass, P. (2013). Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies. Journal of molecular biology, 425(11), 1899-1914.

Figure 3 Binding of hu5B3.v3 and MRCT10.v362 to wild-type and mutant soluble E2 proteins (sE2661) and E2^412–423 peptides.

(a) Ratio of dissociation constants (K_D) for MRCT10.v362 (red) and hu5B3.v3 (black) binding to alanine mutant peptides relative to WT peptide QLINTNGSWHINGSGK-biotin (E2⁴¹²⁻⁴³²-biotin) are graphed. Asterisks denote no binding detected with mutant peptides. (b) Binding of various E2⁴¹²⁻⁴³²-specific neutralizing antibodies to lysates of untransfected 293 cells (No E2) or 293 cells transfected with genotype 2a (J6CF) HCV E1E2-expressing plasmids containing various mutations at N415 or N417. These are representative data from three independent experiments. (c and d) MRCT10 Fab binds to N417S E2⁴¹²⁻⁴³². Biacore sensorgrams demonstrating binding of MRCT10 Fab to WT (c) or N417S (d) E2⁴¹²⁻⁴³² peptide. The Fab series was diluted 2-fold starting from 1000 nM down to 15.6 nM. MRCT10 Fab binds to N417S E2⁴¹²⁻⁴³² peptide with faster off-rate compared to WT. Dissociation constants (K_D) are listed in the graphs.

Pantua, H., Diao, J., Ultsch, M., Hazen, M., Mathieu, M., McCutcheon, K., ... & Hass, P. (2013). Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies. Journal of molecular biology, 425(11), 1899-1914.